The Study Of Bone Marrow Mononuclear Cells Against Ischemic Stroke Through SDF-1/CXCR4 Pathway In Mice

Background and ObjectivesIschemic stroke(cerebral ischemic stroke), also known as cerebral infarction(cerebral infraction), which reduce various blood supply, resulting in local cerebral hypoxic-ischemic tissue necrosis, lead to the neural lineage cell death and relative area neurological dysfunction. Due to the high morbidity, high morbidity and high mortality, ischemic stroke has become the first cause of death and brought a heavy burden on society, it is particularly important to look for effective treatment of cerebral infraction. Although the application of thrombolytic drugs can improve the prognosis, but because of the risk of bleeding and the narrow therapeutic window, which making the majority of patients can not get effective treatment. Currently, the pluripotent stem cell transplantation is the most potential treatment for ischemic stroke. Bone marrow mononuclear cells(BMMNCs) which is composed of a variety ofBone marrow mononuclear cells(BMMNCs) which is composed of a variety of components, including bone marrow mesenchymal stem cells(Mesenchymestemcells, MSCs), endothelial progenitor cells(endothelialpregenitor cells, Ep Cs) and hematopoietic stem / progenitor cell(hematopoieticstem cell, HSCs) and so on. BMMNCs has the advantages of autologous transplantation, easily obtained, without ethical controversy etc, it attracted much attention. The study of ischemic stroke model was found that, the vein graft BMMNCs not only across the BBB(blood brain barrier, BBB) and also can differentiate into neural lineage cells. In ischemic stroke model, many studies shown that transplanted BMMNCs can effectively reduce infarct volume, and promote the recovery of neurological function, but the mechanism is still unclear.Stromal cell-derived factor-1(stromal cell derived factor 1, SDF-1) as α chemoattractant factor family member, which can be expressed in skeletal muscle, bone marrow, heart and brain, and other tissues and organs. When brain tissue ischemia and hypoxia, SDF-1 can recruit stem cells which express CXCR4 receptor to the ischemic area.CXCR4 as a specific receptor, which was widely expressed in MSCs, Ep Cs and HSCs. In the rat model of cerebral ischemia, it was found that SDF-1/CXCR4 pathway can recruit bone marrow MSCs to ischemia brain. AMD3100 as Specific antagonist of SDF- 1, SDF-1 also inhibited the migration of MSCs. Bone marrow MSCs can produce cytokines and act as the relevant cells, and even promote regeneration of damaged tissue, promote the proliferation of endogenous cells, which regulate inflammation and immune response of the host. Because of BMMNCs also can express CXCR4 receptor, it can be inferred that the SDF-1/CXCR4 pathway also plays an important role in BMNNCs treatment of ischemic brain.To explore the neuroprotective mechanism of BMMNCs, this study was designed to assess the safety of BMMNCs transplantation, and investigation whether SDF-1 / CXCR4 pathway plays a role in BMMNCs migration process, which further measurement inflammation and behavioral function in BMMNCs transplanted mice.Part I Tumorigenicity of bone marrow mononuclear cellObjective:The BMMNCs cells were inoculated into nude mice, observing the tumor of nude mice, which assess the security of application.Methods:Twelve enhanced green fluorescent protein(EGFP) which weighing 18-22 g. Twenty four nude mice which weighing 18-22 g. The nude mice were randomly divided into HL-60 group, BMMNCs transplantation group(BMMNCs). The BMMNCs from EGFP mice and HL-60 cells were respectively inoculated subcutaneously into nude mice in each group, and observed that mice living conditions, mental state, diet and defecation. After inoculation cells 60 days, we observed that the general condition of nude mice and the vaccination site of nude mice.Results:Mice were inoculated BMMNCs presence good living conditions, normal diet, good mental stateand normal bowel movement. After inoculation BMMNCs 60 days, nude mice performance good mental state, and the inoculation site no obvious mass formation. Mice which were inoculated HL-60 cells presence the poor mental state and the gradual emergence of cachexia. After inoculation HL-60 60 days, a distinct tumor was found in vaccination site of nude mice.Conclusions:BMMNCs can safely apply in experimental research, and do not have tumorigenicity.Part II SDF-1 / CXCR4 pathway affect migration and differentiation of bone marrow mononuclear cells in ischemic stroke miceObjective:The BMMNCs cells and AMD3100 interventions BMMNCs were transplanted into mice with cerebral infarction, which observed transplanted BMMNCs migration and differentiation in the brain of p MCAO mice, and assess whether the SDF-1 / CXCR4 pathway regulate the migration and differentiation of transplanted BMMNCs in p MCAO mice brain.Methods:Seventy two EGFP mice which weighing 18-22 g. Seventy two male C57/BL6 mice which weighing 18-22 g. C57/BL6 mice were randomly divided into solvent group(Vehicle), BMMNCs transplantation group(BMMNCs) and AMD3100 transplantation group(AMD3100). It applied suture method to create permanent middle cerebral artery occlusion(p MCAO) model. Get EGFP BMMNCs by density gradient method, BMMNCs group and AMD3100 group were separately injected 5 × 106 BMMNCs and 5 × 106 AMD3100 intervention BMMNCs via the tail vein, Vehicle group injected with equal volume of PBS solution. After treatment with BMMNCs, the migration and differentiation was observe at the 7th, 14 th, 28 th, and 35 th day.Results:At the 7th day after BMMNCs transplantation, the number of BMMNCs in BMMNCs group was significantly higher than the number of transplanted BMMNCs in AMD3100 group. Immunofluorescence staining showed that a large number of BMMNCs migrated to infarct area in BMMNCs group, and a handful of EGFP-positive cells co-expressed GFAP and IBA1, and EGFP cells failed to co-expressed Neun, it suggested that transplanted BMMNCs can differentiate into astrocytes and microglia in infarct area, but failed to differentiate into neurons.Conclusions:BMMNCs probably migrate to the cerebral side, and differentiate into microglia and astrocytes in the damaged area through SDF-1 / CXCR4 pathway, which could be the efficacy mechanisms of repair damaged brain tissue.Part III SDF1 / CXCR4 pathway affect vivo factors changes after bone marrow mononuclear cell transplantation in ischemic stroke miceObjective:To study inflammation and nutritional factors after BMMNCs and AMD3100 intervention BMMNCs transplantation to ischemic stroke, which assess the neuroprotective effects of BMMNCs transplant to ischemic stroke mice through SDF-1/CXCR4 pathway.Methods:Forty-eight male EGFP mice, weighing 18-22 g. Forty-eight male C57/BL6 mice, weighing 18-22 g. Experimental animals were randomly divided into sham-operated group(Sham-operated), solvent group(Vehicle), BMMNCs transplantation group(BMMNCs) and AMD3100 transplantation group(AMD3100). Cell transplantation and model-making method were similar with section II. At 3rd days after cell transplantation, Western blot was used to detect the expression of inflammatory cytokines TNF-α, IL-1β, and nutritional factors BNDF, GDNF in mice brain.Results:Western blot quantitative results show that after cell transplantation three days, TNF-α, IL-1β levels of BMMNCs group were significantly lower than AMD3100 group and Vehicle group(P<0.05), and the levels of TNF-α, IL-1β difference between AMD3100 and Vehicle group was not statistically significant(P>0.05). The BNDF and GDNF protein levels of BMMNCs group were significantly higher than AMD3100 group(P<0.05), but the BNDF and GDNF levels difference between AMD3100 group and Vehicle group was not statistically significant(P>0.05).Conclusions:BMMNCs inhibite the expression ofinflammatory cytokines, while promote the expression of trophic factors probably through SDF-1 / CXCR4 pathway, which could be the efficacy mechanisms of repair damaged brain tissue.Part IV SDF-1 / CXCR4 pathway affect cerebral infarct volume and neurological function of bone marrow mononuclear cell transplantation in ischemic stroke miceObjective:To investigate whether SDF1 / CXCR4 pathway affect nerve function and cerebral infarct volume after BMMNCs transplanted in stroke mice, and evaluate BMMNCs therapeutic value for cerebral infarction in mice.Methods:Seventy two male EGFP mice, weighing 18-22 g. Seventy two male C57/BL6 mice weighing 18-22 g. Cell transplantation and modeling methods were similar with section III. At the 1st, 7th, 14 th, 21 th, 28 th, and 35 th day after cell transplantation, which use neurological damage severity score to assess neurobehavioral function.At the 7th day after cell transplantation, which used TTC staining assess infarct volume in ischemic stroke mice.Results:The m NSS scores of BMMNCs group were significantly lower than vehicle group and AMD3100 group, the difference was statistically significant(P<0.05). The difference between BMMNCs group,AMD3100 group and vehicle group did mot show statistically significant after transplantation 1st day(P>0.05), At the 7th, 14 th, 21 th, 28 th, and 35 th day after cell transplantation,the difference between the three groups were statistically significant(P<0.05). TTC staining showed that the total volume of infarction of BMMNCs group was significantly less than Vehicle group and AMD3100 group, the difference was statistically significant(P<0.01).Conclusions:BMMNCs can significantly promote the recovery of nerve function in ischemic stroke mice, and significant reduced infarct volume, which can be as one of the neuroprotective mechanisms.